NOD1^CARD Might Be Using Multiple Interfaces for RIP2-Mediated CARD-CARD Interaction: Insights from Molecular Dynamics Simulation

<div><p>The nucleotide-binding and oligomerization domain (NOD)-containing protein 1 (NOD1) plays the pivotal role in host-pathogen interface of innate immunity and triggers immune signalling pathways for the maturation and release of pro-inflammatory cytokines. Upon the recognition of iE-DAP, NOD1 self-oligomerizes in an ATP-dependent fashion and interacts with adaptor molecule receptor-interacting protein 2 (RIP2) for the propagation of innate immune signalling and initiation of pro-inflammatory immune responses. This interaction (mediated by NOD1 and RIP2) helps in transmitting the downstream signals for the activation of NF-κB signalling pathway, and has been arbitrated by respective caspase-recruitment domains (CARDs). The so-called CARD-CARD interaction still remained contradictory due to inconsistent results. Henceforth, to understand the mode and the nature of the interaction, structural bioinformatics approaches were employed. MD simulation of modelled 1:1 heterodimeric complexes revealed that the type-Ia interface of NOD1^CARD and the type-Ib interface of RIP2^CARD might be the suitable interfaces for the said interaction. Moreover, we perceived three dynamically stable heterotrimeric complexes with an NOD1:RIP2 ratio of 1:2 (two numbers) and 2:1. Out of which, in the first trimeric complex, a type-I NOD1-RIP2 heterodimer was found interacting with an RIP2^CARD using their type-IIa and IIIa interfaces. However, in the second and third heterotrimer, we observed type-I homodimers of NOD1 and RIP2 CARDs were interacting individually with RIP2^CARD and NOD1^CARD (in type-II and type-III interface), respectively. Overall, this study provides structural and dynamic insights into the NOD1-RIP2 oligomer formation, which will be crucial in understanding the molecular basis of NOD1-mediated CARD-CARD interaction in higher and lower eukaryotes.</p></div

Illustration of homodimeric (type-I) mode of interaction.

<p>(A) NOD1^CARD, (B) RIP2^CARD; left panel shows the conserved interactions (acquired from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170232#pone.0170232.s007" target="_blank">S7A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170232#pone.0170232.s008" target="_blank">S8A</a> Figs, respectively) mediated by type-I interfaces of NOD1 and RIP2 CARDs, and the middle panel indicates respective inter-residual/interatomic distances. Detailed 3D representation type-I (1:1 homodimeric) interaction modes were depicted in the right panel. Critical residues involved in intermolecular interactions were shown in ball and stick model, and were coloured based on physicochemical parameters. The black dotted lines represent the inter/intra-molecular polar contacts. The labelled black and red fonts represent the type-Ia and Ib interface residues, respectively.</p

Type-III mode of NOD1-RIP2 interaction.

<p>(A) First type-III interface interaction mode, where type-IIIa interface of NOD1^CARD is found interacting with type-IIIb patch of RIP2^CARD; (B) Second type-III mode of interaction (type-IIIa patch of RIP2^CARD interacts with type-IIIb patch of NOD1^CARD). Left panel of both the figures shows the conserved interactions (taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170232#pone.0170232.s007" target="_blank">S7C</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170232#pone.0170232.s008" target="_blank">S8C</a> Figs), which are mediated by type-III interfaces of NOD1 and RIP2 CARDs; middle panel displays respective inter-residual/interatomic distance graphs. The detailed type-III modes of interactions (along with inter-interface contacts) were depicted in the right panel. The critical residues involved in intermolecular interactions were portrayed in ball and stick model, and were coloured based on physicochemical parameters. NOD1 and RIP2 CARDs models were visualized in white and orange cartoon for better understanding. The black dotted lines represent the inter/intra-molecular polar contacts. The residues labelled in different colour fonts symbolizes the interface types (blue: type-IIa; orange: type-IIb; green: type-IIIa; magenta: type-IIIb).</p

Multiple sequence alignment of NOD1 and RIP2 CARDs.

<p>Sequence alignment of (A) NOD1 and (B) RIP2 CARDs highlights the key residues that drive CARD-CARD interaction. The residues highlighted in different colour bars (based on physicochemical properties) were predicted to be involved in CARD-CARD interaction. The reported and/or predicted residues (from human and zebrafish) are underlined. Coloured circles and triangles (black: type-Ia; red: type-Ib; blue: type-IIa; orange: type-IIb; green: type-IIIa; magenta: type-IIIb) indicate the residues involved in hetero and homodimeric interactions, respectively. GenBank Accessions of NOD1 and RIP2 sequences were considered for multiple sequence analysis (MSA) are as follows: NOD1:- [Human (NP_006083), Chimp (XP_001165528), Monkey (XP_001085719), Mouse (NP_766317), Rat (NP_001102706), Cattle (NP_001243492), Buffalo (XP_006055719), Sheep (XP_011967635), Camel (XP_010986339), Rabbit (XP_002713781), Dog (XP_005628733), Ferret (XP_004762624), Cat (XP_011278751), Mallard (NP_001297310), Chicken (NP_001305367), Zebra finch (XP_012427146), Opossum (XP_001381520), Zebrafish (XP_002665106), Pufferfish (XP_003965935) and Ricefish (XP_011483244)]; RIP2:- [Human (NP_003812), Chimp (XP_519850), Monkey (XP_001084687), Mouse (NP_620402), Rat (NP_001178794), Cattle (NP_001029782), Buffalo (XP_006056644), Sheep (XP_011994115), Camel (XP_010976526), Rabbit (XP_008253935), Dog (XP_005638158), Ferret (XP_004767029), Cat (XP_011289616), Mallard (XP_005029607), Chicken (NP_001026114), Zebra finch (XP_002198519), Opossum (XP_016287297), Zebrafish (NP_919392), Pufferfish (XP_003975835) and Ricefish (XP_004078988)].</p

Complex stability and interatomic/inter-residual distance calculation.

<p>(A) Backbone RMSD of heterotrimeric (NOD1-RIP2-RIP2 and NOD1-RIP2-NOD1) CARD complexes. (B) Principal component analysis of heterotrimeric complexes (all three individual runs; S1, S2, and S3). Upper panel: 2D Projection of first two eigenvectors (PC1 and PC2) in phase space; lower panel: Superimposition of structural coordinates associated with first principal component (PC1). Initial confirmations were coloured in green and the finals were in red and the intermediate structures of NOD1 and RIP2 CARDs were illustrated in silver and orange, respectively. 2D representation of conserved interactions (acquired from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170232#pone.0170232.s004" target="_blank">S4A and S4B Fig</a>) involved in type-II heterodimeric (C) and type-III RIP2-homorimeric interactions (D); the (atomic/residual) distance calculation graphs of all individual interaction were presented in the right panel of each diagram.</p

Structural stability, overview of individual CARDs and the key residues that drive CARD-CARD interaction.

<p>(A) Graphs depict the backbone RMSD and Rg of individual CARDs. (B) Secondary structure components of both NOD1 and RIP2 CARDs during the course of simulation time (the colour segments provided just below the figure represents the secondary structure properties). (C) Cartoon representations of NOD1^CARD and (D) RIP2^CARD displayed the six-helix structural folds (left panel) and electrostatic surface potentials (right panel). The key residues (that were reported or, predicted (in this study) to be involved in CARD-CARD interaction; were presented in sphere-stick model in different colours. The predicted residues are presented in grey and reported residues were portrayed according to their physicochemical properties. The surface view of CARD models shows distinct positive (blue) and negative (red) surface patches. The dotted lines represent the type-I surface patches (blue: type-Ia and red: type-Ib). (E) Multiple sequence alignment of NOD1 and RIP2 CARDs from human (HU), cattle (CA), mouse (MO) and zebrafish (ZF) indicates the key interacting residues. The reported key interacting residues are coloured based on their physicochemical properties.</p

Heterotrimeric complex comparison and interaction analysis.

<p>(A) Comparison of proposed NOD1-RIP2 heterotrimeric complex (left panel: with Ap1-C9; middle: with RIG-I-MAVS; right panel: superimposed view of the said complexes taken from all three individual runs). (B) Illustration of detailed molecular interaction of NOD1-RIP2-RIP2 trimeric CARD complex. The critical amino acids involved in interactions were visualized in ball-stick model and are coloured according to physicochemical parameters. The interface residues were labelled in different colours (black: type-Ia; red: type-Ib; blue: type-IIa; orange: type-IIb; green: type-IIIa; magenta: type-IIIb). 2D representations of heterotrimeric complexes were visualized (in right-upper corner of each illustration) for better understanding of trimeric interface interactions.</p

Analysis of complex stability of heterodimers and total numbers of intermolecular H-bonds formed during the course of simulation.

<p>(A) Graphs represent the backbone RMSD and Rg of NOD1-RIP2 CARD-CARD heterodimeric complexes (analysed from three individual trajectories (S1, S2, and S3)). (B) Principal component analysis of the said complexes. The projection graphs of first two eigenvectors (x-axis represents the PC1 and y-axis symbolizes PC2) of individual CARD-CARD complexes (type-I mode) indicate the global motion of complexes in phase space (upper panel). In lower panel, the superimposition of structural coordinates associated with the principal component 1 (PC1) of NOD1-RIP2 complexes display the global motion. The initial conformations were colour in blue, the final in red and the intermediate ones were displayed in silver (NOD1^CARD) and orange (RIP2^CARD). (C) The graphs indicate total number of H-bonds that formed by individual CARD-CARD complexes (type-I mode) during 50ns MD simulation in all three individual production runs.</p

Variation of H-bonds participated in interaction during 50 ns simulation in zNOD-NACHT complexes.

<p>(A) Complex I for zNOD1-NACHT, (B) Complex II for zNOD1-NACHT, (C) Complex III for zNOD1-NACHT, (D) Complex I for zNOD2-NACHT, (E) Complex II for zNOD2-NACHT and (F) Complex III for zNOD2-NACHT (G) mNLRC4-NACHT complex. The y-axis represents the number of H-bond formed during the course of MD simulation and the simulation time (in ns) is depicted in x-axis. All graphs are generates using Grace 5.1.23 plotting program.</p

Interaction analysis (hydrogen bond, hydrophobic contacts, electrostatics and van der Waals contact) of NOD1-NACHT-ATP and NOD2-NACHT-ATP complexes before molecular dynamics simulation.

<p>Interaction analysis (hydrogen bond, hydrophobic contacts, electrostatics and van der Waals contact) of NOD1-NACHT-ATP and NOD2-NACHT-ATP complexes before molecular dynamics simulation.</p